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- Title
659. Application of HSVtk Suicide Gene to X- SCID Gene Therapy: Ganciclovir Treatment Selectively Elmiinates Gene Corrected Cells In Vitro and In Vivo.
- Authors
Uchiyama, Toru; Ishikawa, Yoshinori; Ishii, Naoto; Onodera, Masafumi; Sato, Miki; Sasahara, Yoji; Tanaka, Nobuyuki; Sugamura, Kazuo; Kumaki, Satoru; Tsuchiya, Shigeru
- Abstract
X-SCID is characterized by profound defects of humoral and cellular immunity due to mutations in the gene encoding the common gamma chain (γc). Recent clinical trials of X-SCID gene therapy demonstrated the successful immunological reconstitution in the patients. However, a serious adverse effect of uncontrolled clonal T cell proliferation due to insertional mutagenesis of retroviral vector was reported.To offset the side effect, we applied the suicide gene system to X-SCID gene therapy as a fail-safe measure in case uncontrolled proliferation of gene transduced lymphocytes occurred. For this purpose, we constructed a bicistronic retroviral vector, pD-γc/TK, carrying human γc cDNA and HSVtk suicide gene. In the present study, MSCV LTR-based vector was used to overcome gene silencing, because a long-term reduction of the γc expression had been observed under the control of MoMLV LTR-based vector in our previous study. First, we transduced B-cell lines from X-SCID patients with this vector. Expression of the γc was stable for more than six months. After confirmation of functional reconstitution of the γc, the cells were treated with various concentrations of ganciclovir (GCV). The γc positive cells were selectively eliminated under lower concentrations of GCV (<25μM), and have not reappeared at least for 6 months. Furthermore, the γc transduced cells were still sensitive to GCV after six months. These results demonstrate that pD-γc/TK maintains a long-term simultaneous expression of the γc and HSVtk genes.To test the efficacy and safety feature of the gene therapy in vivo, we utilized pD-γc/TK for transduction of X-SCID murine model. Bone marrow cells obtained from X-SCID mice were prestimulated with growth factors including mSCF, mIL-11 and mTPO for 48hr and then transduced with the retroviral supernatant. The transduced cells were harvested and injected into lethally irradiated (800 rad) X-SCID mice. Two months after transduction, peripheral blood cells from the transduced mice were analyzed. Before transduction, the X-SCID mice show a decrease in the absolute number of lymphocytes comprising of a reduced numbers of B cells and CD8+ T cells with a relative accumulation of CD4+ T cells. Transfer of the γc gene resulted in an increase in CD8+T cells and B cells with a substantially corrected CD4:CD8 T-cell ratio. In addition, the γc expression was detected on the populations of these restored lymphocytes. These restored lymphocytes could be eliminated by 50 mg/kg GCV when injected into peritoneal for seven consecutive days. However, this treatment caused acute toxicity to the injected mice. Currently, we are optimizing the concentration of GCV, which could selectively eliminates the gene transduced lymphocytes without obvious toxicity in vivo.Molecular Therapy (2006) 13, S254–S254; doi: 10.1016/j.ymthe.2006.08.736
- Subjects
SEVERE combined immunodeficiency; GENETIC engineering; GENE therapy; T cells; BONE marrow cells
- Publication
Molecular Therapy, 2006, Vol 13, pS254
- ISSN
1525-0016
- Publication type
Article
- DOI
10.1016/j.ymthe.2006.08.736